Difference between revisions of "Team:WashU StLouis/Proof"
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<img src = "https://static.igem.org/mediawiki/2016/b/b2/T--WashU_StLouis--BiotinSynthesis.gif" style="display: block; margin: 0 auto; width: 400px;float:right;"> | <img src = "https://static.igem.org/mediawiki/2016/b/b2/T--WashU_StLouis--BiotinSynthesis.gif" style="display: block; margin: 0 auto; width: 400px;float:right;"> | ||
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<p> | <p> | ||
After identifying our genes of interest and constructing the electron donor overproducing plasmids, we performed an assay in order to see if our new cells produced usable forms of reduced electron donors. Our assay showed that not only were the products were overproduced, but also significant data supported the claim that these electron donors were usable in protein synthesis. | After identifying our genes of interest and constructing the electron donor overproducing plasmids, we performed an assay in order to see if our new cells produced usable forms of reduced electron donors. Our assay showed that not only were the products were overproduced, but also significant data supported the claim that these electron donors were usable in protein synthesis. | ||
| + | </p>--> | ||
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| + | <p>After analyzing the results of our absorbance data of electron donors, we decided to go a step further to ensure that our electron donor constructs were producing reduced and usable electron donors. We performed an assay which showed that not only were the products being overproduced, but also that there was significant data supporting the claim that these electron donors were usable in protein synthesis. | ||
</p> | </p> | ||
<p> | <p> | ||
| − | + | Biotin, commonly know as vitamin B7, is produced by a pathway that uses reduced flavodoxin/ferredoxin,specifically the SAM pathway that turns dethiobiotin to biotin[1] (see diagram below). Specifically, there is a 1:1 stoichiometric relationship between reduced electron donors and biotin molecules produced. If we measure the amount of biotin in our cells, we will indirectly measure the amount of reduced electron donors present in our cells. We used a readily available biotin assay to conduct our proof of concept experiment. | |
</p> | </p> | ||
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<p> | <p> | ||
| − | + | Results showed that overall, our constructs worked to increase biotin production, as seen in the graphs below. | |
<ul> | <ul> | ||
<li>When our constructs were in DH10B cells, the <i>fldA</i>, <i>fldA</i>-<i>pfo</i>, <i>petF</i>, and <i>petF</i>-<i>pfo</i> strains produced more biotin than the control DH10B strain at some induction level</li> | <li>When our constructs were in DH10B cells, the <i>fldA</i>, <i>fldA</i>-<i>pfo</i>, <i>petF</i>, and <i>petF</i>-<i>pfo</i> strains produced more biotin than the control DH10B strain at some induction level</li> | ||
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<img src = "https://static.igem.org/mediawiki/2016/c/c5/T--WashU_StLouis--BiotinYalegraph.png" style="width : 40vw;display: inline-block; "> | <img src = "https://static.igem.org/mediawiki/2016/c/c5/T--WashU_StLouis--BiotinYalegraph.png" style="width : 40vw;display: inline-block; "> | ||
</div> | </div> | ||
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| + | <p>This experiment illustrates a proof of concept for our Super Cells. By showing that our cells could overproduce biotin, we have shown that the theory behind the Super Cells work: that overproducing certain co-factors will increase the amount of resulting product<p> | ||
<p>Go to the <a herf="https://2016.igem.org/Team:WashU_StLouis/Conclusions">conclusions page</a> to see a complete summary of our results and proof of concept experiments.</p> | <p>Go to the <a herf="https://2016.igem.org/Team:WashU_StLouis/Conclusions">conclusions page</a> to see a complete summary of our results and proof of concept experiments.</p> | ||
Revision as of 22:21, 18 October 2016
Proof of Concept
Measuring biotin production to calculate reduced electron donors
After analyzing the results of our absorbance data of electron donors, we decided to go a step further to ensure that our electron donor constructs were producing reduced and usable electron donors. We performed an assay which showed that not only were the products being overproduced, but also that there was significant data supporting the claim that these electron donors were usable in protein synthesis.
Biotin, commonly know as vitamin B7, is produced by a pathway that uses reduced flavodoxin/ferredoxin,specifically the SAM pathway that turns dethiobiotin to biotin[1] (see diagram below). Specifically, there is a 1:1 stoichiometric relationship between reduced electron donors and biotin molecules produced. If we measure the amount of biotin in our cells, we will indirectly measure the amount of reduced electron donors present in our cells. We used a readily available biotin assay to conduct our proof of concept experiment.
Results showed that overall, our constructs worked to increase biotin production, as seen in the graphs below.
- When our constructs were in DH10B cells, the fldA, fldA-pfo, petF, and petF-pfo strains produced more biotin than the control DH10B strain at some induction level
- When our constructs were in the ΔaceE knockout cells, the fldA, fldA-pfo, and petF strains all produced more biotin than the control DH10B strain, control ΔaceE knockout strain, and DH10B+constructs strain (petF-pfo data is lacking due to its inability to grow)
This experiment illustrates a proof of concept for our Super Cells. By showing that our cells could overproduce biotin, we have shown that the theory behind the Super Cells work: that overproducing certain co-factors will increase the amount of resulting product
Go to the conclusions page to see a complete summary of our results and proof of concept experiments.
References
- Lin, Steven, and John E. Cronan. "Closing in on complete pathways of biotin biosynthesis." Molecular BioSystems 7.6 (2011): 1811-1821.
- Picciocchi, Antoine, Roland Douce, and Claude Alban. "Biochemical characterization of the Arabidopsis biotin synthase reaction. The importance of mitochondria in biotin synthesis." Plant physiology 127.3 (2001): 1224-1233.
